JP2000159669A - Urease inhibitor, medicine and food composition containing the inhibitor and measurement of urease activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a urease inhibitor having high safety, a medicine containing the inhibitor as an active ingredient, a food additive and a food composition containing the urease inhibitor and to provide a method capable of measuring urease activity even in the presence of a compound having a phenol structure. SOLUTION: This urease inhibitor contains proanthocyanidin or its salt. This medicine contains the urease inhibitor as an active ingredient. This food additive and this food composition contain the urease inhibitor. This method for measuring urease activity comprises forming ammonia by hydrolyzing urea by urease, reacting the formed ammonia with dansyl chloride to form dansylamide and determining formed dansylamide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a urease inhibitor, a pharmaceutical and food composition containing the same, and a method for measuring urease activity. The urease inhibitor of the present invention includes Helicobactor pylori:
Hereinafter, it is simply referred to as “pylori”. ) Etc. can significantly inhibit urease activity. This means that according to the urease inhibitor of the present invention, in the digestive tract, particularly in the stomach, it is possible to inhibit the growth of H. pylori in the digestive tract by suppressing the production of ammonia caused by H. pylori infection. is there. Thus, the urease inhibitor of the present invention is extremely useful for treating or preventing gastritis, gastric ulcer, gastric cancer and the like caused by H. pylori.

[0002]

BACKGROUND OF THE INVENTION At present, one of the most prevalent diseases is peptic ulcer. In recent years, a large part of the stomach ulcer with the advent of histamine H ₂ receptor antagonists, peptic ulcers such as duodenal ulcers without the need for surgical treatment, at least say was I came to healing. However, many of the patients who had been cured with this treatment were found to recur repeatedly, and the cause was determined. As a result, H. pylori, a kind of helical bacteria, was detected in the majority of stomachs of relapsed patients, and the relationship between H. pylori, gastric ulcer, gastritis, and gastric cancer was clarified epidemiologically (for example, June 21, 1998). An article reported by the Mainichi Shimbun of the Japan Gastric Cancer Society held in Tokyo on the same month said that "Helicobacter pylori, a bacterium that causes stomach ulcer and duodenal ulcer, has the function of causing gastric cancer. (Abbreviation) Infection with H. pylori promotes carcinogenesis by carcinogens (abbreviation). "

[0003] Here, H. pylori holds urease, which accounts for as much as 6% of the cell weight, decomposes urea present in gastric juice to produce ammonia, and passes through the acidic environment inside the cells and around the cells. By harmony, it enables its inhabitation in the stomach. H. pylori, on the other hand, secretes cytotoxins,
This cytotoxin produces active oxygen from neutrophils directly or via interleukin-8, and finally produces extremely cytotoxic monochloramine, which damages the gastric mucosa and the duodenal mucosa and causes the gastric ulcer. Causes gastritis, gastric cancer, and duodenal ulcer. The effect of these pylori on the mucous membrane is schematically shown in FIG. “SOD” in FIG. 1 indicates superoxide dismutase.

Accordingly, in order to cure and prevent or prevent peptic ulcer and gastric cancer from recurring, it is necessary to prevent pylori from inhabiting it, which inhibits the urease activity of pylori and neutralizes acidity. The method of suppressing the production of ammonia for the purpose is effective. As antiulcer agents produced from this viewpoint, omeprazole, which is a proton pump inhibitor having urease inhibitory activity, and several therapeutic agents having chemical structures similar thereto are known.

[0005]

However, the proton pump inhibitors described above exhibit weak basicity due to their chemical structure, and are therefore specifically stored in hydrochloric acid-secreting cells for a longer period than necessary. Therefore, it is feared that these therapeutic agents continue to inhibit acid secretion from cells for a long period of time and also have a side effect on bone metabolism and the like. In this case, its use is only approved for acute ulcers.

Accordingly, an object of the present invention is to provide a highly safe urease inhibitor having no such undesirable effects. Another object of the present invention is to provide a medicament containing the urease inhibitor as an active ingredient, and a food additive or a food composition containing the urease inhibitor. These urease-inhibiting active agents, medicaments, food additives and food compositions of the present invention have significant inhibitory activity on the urease activity of H. pylori, and suppress the growth of H. pylori in the stomach or remove bacteria. Can be suitably used.

Another object of the present invention is to provide a method for measuring urease, which can effectively measure urease activity even in the presence of a compound having a phenol structure. As a method for measuring the activity of urease, the so-called indophenol method shown in FIG. 2 has been conventionally used. In other words, the method includes steps 4 to
By measuring the blue absorbance of indophenol resulting from the two reaction steps, the amount of ammonia produced in the first step I (the amount of hydrolysis of urea to ammonia by urease) is determined, and the urease activity is measured. is there. However, according to this method,
Since salicylic acid having a phenol structure is used in Steps III and IV, the presence of another phenol compound in this reaction system causes a problem that a large error is caused. For example, urease of a compound having a phenol structure as in the present invention is used. It could not be used as a measuring method for evaluating inhibitory activity.

[0008]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and found that proanthocyanidin, which is present in many natural edible plants such as grape berries, has an urease inhibitory activity. In particular, it was confirmed that the action was significant for a crude product of proanthocyanidin extracted and prepared from a natural product, preferably for a composition further containing catechins.
Furthermore, based on such findings, the present inventors further demonstrated that the above proanthocyanidins or the crude product thereof significantly inhibited the urease activity of H. pylori and proliferated H. pylori present in the stomach by ingesting H. pylori-infected patients. Was confirmed, and it was confirmed that the bacteria could be removed. The present inventors have also found that oral administration of proanthocyanidin and the like can significantly treat and prevent gastric ulcer. The present invention has been developed based on such knowledge.

That is, the present invention is a urease inhibitor containing proanthocyanidin.
The urease inhibitor includes the following embodiments. (1) The urease inhibitor described above, which further contains catechins. (2) The urease inhibitor containing proanthocyanidin in an amount of 60 to 95% by weight and catechins in an amount of 5 to 40% by weight. (3) The above urease inhibitor, wherein the proanthocyanidin is derived from French pine or grape seed. The present invention is also a medicament comprising the above urease inhibitor as an active ingredient. The medicament includes both those having the activity of inhibiting the growth or eradication of H. pylori and used for the purpose, and those having the anti-ulcer activity and used for the purpose. Furthermore, the present invention is a food additive containing the above urease inhibitor. The food additive includes both those having an inhibitory effect on the growth or elimination of H. pylori and used for the purpose, and those having an anti-ulcer effect and used for the purpose. Still further, the present invention is a food composition containing proanthocyanidin, which has a growth inhibitory property against bacteria and a disinfecting property against H. pylori, and is used for the purpose. The food composition may further contain catechins. Furthermore, the present invention is a food composition containing proanthocyanidins, which has antiulcer properties and is used for that purpose. The food composition may further contain catechins.

Further, the present inventors have conducted intensive studies on a method capable of effectively evaluating and measuring urease activity even in the presence of a compound having a phenol structure. As a result, it was found that urease hydrolyzes urea to produce ammonia. By reacting with dansyl chloride to form dansylamide, and quantifying this, it was found that the amount of hydrolysis of urea to ammonia by urease, ie, urease activity, could be quantitatively measured.
That is, the present invention provides a method for measuring urease activity, which comprises producing ammonia by hydrolyzing urea with urease, and then quantifying dansylamide produced by reacting the produced ammonia with dansyl chloride. is there. In this specification and the drawings, “%” representing a concentration refers to% by weight, unless otherwise specified.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The urease inhibitor of the present invention has the following formula (I):

[0012]

Embedded image

(Wherein R is the same or different and represents H or OH, and X represents an integer of 2 or more) (hereinafter, also referred to as “chemical compound I”). ). R in the formula (I) may be the same or different and may be any of H and OH, and does not particularly matter whether it is H or OH. For example, all Rs in the formula (I) may be either H or OH, or a part of Rs in the formula (I) is either H or OH and the remainder of R May be the other. The repeating number X in the formula (I) is not particularly limited as long as it is an integer of 2 or more, but is usually an integer in the range of 2 to 20. These R and X vary depending on the origin of the acquisition and the preparation method, and the present invention includes any of those obtained according to the origin of the acquisition or the preparation method.

As proanthocyanidins, specifically, condensed tannins contained in plants, ie, flavan-3
A condensed compound or a polymerized compound having -ol or flavan-3,4-diol as a basic unit;
More specifically, properalgonidi (properalgonidi)
n), procyanidin (Prosyanidin), prodelphinidin (Prodekfinidin), progibortinidin (Proguib)
ourtinidin), profisetinidin (Prosetinidin),
Prorobinetinidin, Proteracacidin, Promelacacidin
acacidin), proapigeninidi
n), proanthocyanidins such as proluteolinidin and stereoisomers thereof. The urease inhibitor of the present invention may contain one type of proanthocyanidin, or may contain two or more types of proanthocyanidins.

Although proanthocyanidins may be produced by various methods, they are generally contained in a large amount of plants such as trees and fruits, and are therefore preferably produced by extracting and isolating from these plants. By using proanthocyanidins extracted and isolated from natural products such as plants in this manner, there is no problem that can be caused by toxic side reactants generated when producing using synthetic reactions and the like. It can be manufactured at low cost. Various plants can be used without particular limitation for extracting and isolating (preparing) proanthocyanidins. For example, woody parts (bark), fruits (epidermis), peanuts containing a large amount of proanthocyanidins can be used. (Skin), seeds (skin), leaves and flowers of trees (trees and shrubs) and the like may be used, preferably pine or grape can be suitably used, and more preferably pine bark or grape Seeds can be used. In the case of using pine, it is particularly preferable to use pine on the French coast.

The method of preparing proanthocyanidins from natural products such as these plants is not particularly limited and can be carried out according to known methods. For example, JP-A-8-225
No. 453, page 3, column 4, line 3 to line 46, discloses a method for extracting and purifying compound I from a plant with a solvent. Thus, a composition containing compound I is prepared using this method. Can be prepared. In addition, if necessary, in order to increase the purity of proanthocyanidin, as described in the same publication, it is further purified by subjecting it to, for example, an adsorption treatment by adsorption column chromatography, a membrane separation treatment, a solvent separation treatment, or the like. You may. The composition containing proanthocyanidin thus obtained is often a mixture of various R and X in the formula (I),
In the urease inhibitor of the present invention, a mixture thereof is suitably used. The urease inhibitor of the present invention desirably contains a crudely purified proanthocyanidin extracted and isolated from a plant as described above. As a crudely purified proanthocyanidin, proanthocyanidin is at least about 50%, preferably 6%.
A composition containing 0 to 70% may be mentioned. The other components are not particularly limited, but preferably include catechins. Therefore, the urease inhibitor of the present invention preferably contains proanthocyanidins and catechins. The mixing ratio of these is not particularly limited, but the proanthocyanidins 60 to 95%
And catechins 5 to 40%, preferably 70 to 90% proanthocyanidins and 10 to 30% catechins. The catechins to be added to the urease inhibitor of the present invention are not particularly limited,
Examples include catechins such as (+)-catechin, dl-catechin, (+)-epicatechin, epigallocatechin, epicatechin, and various isomers thereof. The catechins to be added to the urease inhibitor of the present invention may be composed of one of the above catechins,
It may be a combination of more than one kind. Preferably, the combination of catechin and epicatechin is good, and more preferably, the combination of catechin. A composition containing proanthocyanidins and catechins,
As described above, it can be obtained by extracting and isolating from a plant, but a commercially available product can be conveniently used, and such a commercially available product is not limited.
(Product name, Glory International Inc.)
Can be exemplified.

The urease inhibitor of the present invention can be used for various purposes. For example, the urease inhibitor of the present invention is used as an active ingredient of a medicament used for treating or preventing a disease caused by the action of urease,
It can also be used as a food additive in the preparation of food. As described above, H. pylori degrades urea present in gastric juice by its own urease to produce ammonia, neutralizing the acidic environment around bacteria and enabling inhabitation in the stomach, It secretes cytotoxins and damages the gastric mucosa and duodenum. A pharmaceutical composition containing the urease inhibitor, a food additive or a food composition containing the same,
When used orally, it inhibits the inoculation of H. pylori in the stomach on the basis of its urease inhibitory activity, thereby suppressing its proliferation and eventually eliminating it, and also stomach ulcers Can be useful for the treatment or prevention of Therefore, the present invention provides a medicine containing the above-mentioned urease inhibitor as an active ingredient. That is, the medicament of the present invention contains the above-mentioned proanthocyanidin as an active ingredient, and can further contain the above-mentioned catechins. Preferably, it contains both proanthocyanidins and catechins as active ingredients, and the mixing ratio of these is not limited, but as described above, 60 to 95% of proanthocyanidins and 5 to 40% of catechins, preferably Anthocyanidin 70
-90% and catechins 10-30%.

The medicament of the present invention comprises, in addition to the above-mentioned active ingredients,
Depending on the purpose of administration, various pharmaceutically acceptable carriers, additives and the like can be appropriately blended to prepare various formulations. Pharmaceutically acceptable carriers include diluents or excipients, such as fillers, extenders, binders, humectants, disintegrants, surfactants, and lubricants, which are commonly used, depending on the use form of the preparation. Excipients can be exemplified, and these are appropriately selected and used depending on the dosage unit form of the preparation. The administration form of the medicament of the present invention is not particularly limited, but typical examples thereof include tablets, pills, powders, fine granules, granules, capsules, syrups, solutions, suspensions, emulsions and the like. Oral preparations can be mentioned. Preferred are tablets, granules, capsules and liquids.

For example, in the case of molding into a tablet form, the above-mentioned preparation carrier such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. Agents: water, ethanol, propanol, simple syrup,
Binders such as glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone: sodium carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried starch, sodium alginate, agar powder, laminaran Powder, disintegrators such as sodium hydrogen carbonate and calcium carbonate: surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, and stearic acid monoglyceride: disintegrators such as sucrose, stearin, cocoa butter, and hydrogenated oil: Glycerin,
Moisturizers such as starch: adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid: purified talc,
Lubricants such as stearates, boric acid powder, and polyethylene glycol can be used. For example, when formed into a pill form, as a pharmaceutical carrier, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, etc .: binding of gum arabic powder, powder of tragacanth, gelatin, ethanol, etc. Agents: Disintegrators such as laminaran and agar can be used. Further, the pharmaceutical preparation of the present invention may contain various additives such as a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent, and other medicines, if necessary. The amount of the urease inhibitor of the present invention contained in the pharmaceutical preparation of the present invention is not particularly limited and may be appropriately selected from a wide range. However, the amount of proanthocyanidin usually ranges from about 10 to 50% in 100% of the pharmaceutical preparation. Can be mentioned.

The medicament of the present invention can also inhibit the urease activity of H. pylori based on the use of a urease inhibitor as an active ingredient, thereby inhibiting or eliminating the growth of H. pylori present in the gastrointestinal tract such as the stomach. It can be suitably used for the purpose of bacteria, that is, as a growth inhibitor or a disinfectant for H. pylori. Further, the medicament of the present invention can be suitably used for the purpose of anti-ulcer for treating or preventing gastrointestinal ulcers such as gastric ulcer and duodenal ulcer caused by pylori, that is, as an anti-ulcer agent.

The dosage of the pharmaceutical preparation is not particularly limited as long as the desired therapeutic or prophylactic effect can be obtained. Usually, the dosage, age, sex and other conditions of the patient, the type of the disease, and the It is appropriately selected according to the degree and the like. Usually, the amount (dry weight) of the proanthocyanidin as an active ingredient is preferably about 1 to 10 mg / kg per day, which can be administered in 1 to 4 times a day.

Further, the present invention provides a food additive containing the above-mentioned urease inhibitor as an active ingredient. The food additive of the present invention contains proanthocyanidin as an active ingredient similarly to the above-mentioned medicines, and can further contain the above-mentioned catechins. Preferably, it contains both proanthocyanidins and catechins as active ingredients. The mixing ratio of these is not limited, but as described above, 60 to 95% of proanthocyanidins and 5 to 40% of catechins, preferably
0-90% and catechins 10-30%. The food additive of the present invention may contain, in addition to the urease inhibitor, which is an active ingredient, various additives such as flavors, flavors, and stabilizers widely used in the food field. The food additive of the present invention is used for the purpose of inhibiting the growth or eradication of pylori present in the gastrointestinal tract such as the stomach, or for improving or preventing gastrointestinal ulcers such as gastric ulcer and duodenal ulcer caused by pylori. It is used by adding it to various foods for the purpose of. The type of the target food is not particularly limited, and can be applied to any food. For example, soft drinks (juices, sports drinks, milk drinks, tea drinks, coffee drinks, carbonated drinks, etc.), milk, sweets (chocolate, candy, gum, cookies, biscuits, snacks, etc.), ice desserts (ice) Cream, popsicles, sherbet, etc.). The amount of the food additive of the present invention used in the food is not particularly limited as long as the food is ingested and exerts a growth inhibitory effect or a disinfecting effect of H. pylori, or an antitumor effect. It is appropriately selected and used in consideration of the target intake and the like. Specifically, the daily intake of proanthocyanidins is 1 to 10 mg / kg, preferably 1 to 10 mg / kg.
It is appropriately selected and used so as to be 5 mg / kg.

Further, the present invention is a food composition containing proanthocyanidins. This food composition is based on the urease inhibitory activity of proanthocyanidins, for the purpose of inhibiting the growth or elimination of H. pylori in vivo,
Alternatively, it can be suitably used on the day of antiulcer. These food compositions may further contain catechins. The mixing ratio of these proanthocyanidins and catechins is not limited, but, for example, 5-67% of catechins with respect to 100% of proanthocyanidins, preferably 11-43% of catechins with respect to 100% of proanthocyanidins. be able to.

The food composition of the present invention is prepared by mixing proanthocyanidin or catechins with a carrier such as a filler or excipient which is acceptable for food hygiene, in the form of solids such as tablets and pills, or syrups. Or proanthocyanidins or catechins, for example, soft drinks (juices, sports drinks, milk drinks, tea drinks, coffee drinks, carbonated drinks, etc.), milk, confectionery (Chocolate, candy, gum, cookies, biscuits, snacks, etc.) and ice desserts (ice cream, ice lolly, sherbet, etc.). Since proanthocyanidins are substances that are also contained in edible plants, there is no safety issue in using them as components of food compositions. Even when the food composition of the present invention contains catechins, there is no problem in safety because catechins are also components contained in edible plants such as green tea. If the concentration of the proanthocyanidins or catechins in the food composition is too high, the taste of the food composition tends to be impaired.If the concentration is too low, the purpose is to suppress the growth or eradication of pylori or to prevent ulcers caused by pylori. Since the purpose of the ulcer cannot be achieved at a sufficient level, it is preferable to set the range to satisfy both of these conditions. Usually, it is preferably 0.05 to 0.2% per 100% of the food composition of the present invention.

Further, the food composition of the present invention containing proanthocyanidins or catechins in addition has anti-proliferative or eradication properties against H. pylori and anti-ulcer properties against ulcers caused by H. pylori. Can be used for the purpose. The food composition can be used as a functional food, a health food, a health-conscious food, and the like. The food composition is divided into several times a day, and the proanthocyanidin intake per day is 1 to 10 mg / kg, preferably 1 to 5 mg / kg.
It is preferable to be ingested so as to be mg / kg.

The present invention further provides a novel method for measuring urease activity. FIG. 3 shows the principle of the urease activity measurement method of the present invention. As can be seen from FIG. 3, the method for measuring urease activity of the present invention comprises reacting urease with urea (step 1), and then reacting ammonia produced by the hydrolysis of urea with dansyl chloride to produce dansyl amide. This is a method of measuring the urease activity of urease by quantifying the produced dansylamide (Step 3). The reaction method and reaction conditions of Step 1 can be performed according to Step I of the conventional indophenol method,
For example, the conditions include a temperature of about 37 ° C. and a pH of about 7. The reaction of Step 2 is not limited, but can be usually performed at a temperature of 30 ° C to 40 ° C, preferably about 37 ° C, and a pH of 10 to 12.5, preferably pH 11 to 12. The amount of dansyl chloride to be used is not particularly limited as long as the amount of the ammonia generated in Step 1 is almost completely reacted. However, usually, the amount of the dansyl chloride is a large excess equivalent to the ammonia. Unreacted dansyl chloride is removed by washing with water after reacting with an amino acid (such as glycine). Various methods may be used for the quantification of the generated dansylamide (Step 3). For example, it can be performed using the fluorescence of dansylamide. In this sense, the method for measuring urease activity of the present invention can also be referred to as a fluorescent labeling method. Conveniently, for example, high performance liquid chromatography (HPL) equipped with a fluorescence detection device
The quantification of dansylamide can be easily performed by C) or thin layer chromatography. Particularly preferred is high performance liquid chromatography, which allows easy and accurate quantification. Unlike the conventional indophenol method, the fluorescent labeling method of the present invention can be performed even under conditions where a compound having a phenol structure coexists in the reaction system. Therefore, for example, it is suitable for measuring the urease inhibitory activity of a compound having a phenol structure, and is useful for screening and evaluating such a compound. Needless to say, it can be suitably used for evaluating and measuring the urease inhibitory activity of other compounds. Furthermore, in the conventional indophenol method, if DMSO (dimethylsulfoxide), which is frequently used as a solubilizing agent, is present in the reaction system, there is a problem that color interference occurs and the urease inhibitory activity cannot be measured. According to the labeling method, since such a problem caused by DMSO does not occur, the urease activity or the urease inhibitory activity can be evaluated and measured even when DMSO is present in the reaction system.

[0027]

EXAMPLES Examples and test examples will be given below to specifically explain the present invention. However, the present invention is not limited by the embodiments and the like.

Example 1 a) Urease Inhibitory Activity The urease inhibitor of the present invention was evaluated for its urease inhibitory activity by determining the 50% inhibitory concentration (IC ₅₀ value) of urease inhibitor of the present invention with respect to urease. As the urease inhibitor of the present invention, "OPC" (trade name) provided by Glory International Co., Ltd. was used. FIG. 4 shows the result of composition analysis of the “OPC” by liquid chromatography. As you can see from the figure, "OPC"
Contained about 65% proanthocyanidins and about 30% catechins (catechin and epicatechin). As a control urease inhibitor, acetohydroxamic acid (Acetohyd
roxamic acid) and omeprazole (positive control). FIG. 5 shows the chemical structural formulas of acetohydroxamic acid and omeprazole. Specifically, the procedure was performed as follows. Urease (2.4 units / ml, 0.1 M phosphate buffer (pH 7.0)) derived from common bean (Jack Bean) was used as the urease, and 100 μl of the urease (0.12 units / final concentration) was used.
ml), 50 μl of each urease inhibitor solution prepared to various concentrations with 1% DMSO and 650 μl of 0.1 M phosphate buffer (pH 7.0) were added. In addition,
The 0.1 M phosphate buffer (pH 7.0) is prepared by adding a 0.1 M sodium phosphate solution to a 0.1 M disodium phosphate solution to adjust the pH to 7.0 (using water for injection). .

Thereafter, the reaction was carried out at 37 ° C. for 10 minutes, and 100 μl of a 2.0 mg / dl urea solution (0.1 M phosphate buffer, pH 7.0) (final concentration: 0.1 mg / dl) was added to each reaction solution.
ml), and the mixture was further reacted at 37 ° C. for 15 minutes. Next, 500 μl of a 10% by weight aqueous solution of potassium carbonate (using water for injection) and dansyl chloride (D
NS-CI) and 600 μl of a 0.5% by weight acetone solution. Thereafter, the reaction was carried out at 55 ° C. for 90 minutes, 3 ml of a 5% aqueous solution of glycine (using water for injection) was added to each reaction solution, and the reaction was further carried out at 55 ° C. for 30 minutes. Thereafter, the reaction solution was cooled with ice water for 3 minutes, 5 ml of water for injection was added to each reaction solution, and the reaction solution was allowed to cool to room temperature. 2 ml of chloroform was added to each cooled reaction solution, and the mixture was stirred and filtered with a liquid phase separation filter paper to obtain an organic (chloroform) layer. Thereafter, the solvent was distilled off from the obtained organic layer with nitrogen gas. Internal standard substance (DNS-dimethylamide 1 ×
10 ^{- 6} _CH 3 CN1ml plus M), was added to the evaporated organic layer, the thus obtained mixture YM
C Disposable Filtration Filter Duo-Filt
er QDUO04 (trademark of YMC Corporation)
And filtered.

The obtained filtrate was analyzed by high performance liquid chromatography (HPLC) to quantify dansylamide. The analysis conditions of the high performance liquid chromatography used are as follows. (Column) YMC-Pack ODS-A, S-5μ
m, 120Å 150 × 6.0 mm I.m. D. (Mobile phase) Acetonitrile: 17.5 mM phosphate buffer (pH 7.2) = 1: 1, and 17.5 mM phosphate buffer (pH 7.2) was added to 17.5 mM dipotassium phosphate solution. Add 5 mM mono-potassium phosphate solution and adjust pH
Adjusted to 7.2 (using water for injection). (Flow rate) 1 ml / min (Fluorescence detection wavelength) EX 340 nm, EM 530 nm (Temperature) 30 ° C. When eluted under these conditions, a peak of dansylamide appeared at about 7.8 minutes (see FIG. 3). Dansylamide was quantified from the peak area. The urease activity inhibition of each inhibitor was then evaluated, the concentration of each inhibitor giving 50% inhibition was determined, and the IC ₅₀ value (50% inhibitory concentration) was determined. Table 1 shows the results.

[0031]

Table 1 Table 1 IC _{50 of} each urease inhibitor (50%
Inhibition concentration) Sample name IC ₅₀ Acetohydroxamic acid 1.5 μg / ml Omeprazole 0.5 μg / ml Urease inhibitor of the present invention 0.47 μg / ml

As shown in Table 1, the urease inhibitor of the present invention inhibits urease at 50% of the concentration of about one third of acetohydroxamic acid, a typical urease inhibitor, and is almost equivalent to omeprazole. The concentration was found to inhibit urease by 50%. This proved that the urease inhibitor comprising the proanthocyanidin of the present invention as an active ingredient had an extremely high urease inhibitory activity.

B) Comparison of urease inhibitory activity between urease inhibitor of the present invention and catechins Using the method used in a) above, the urease inhibitory activity of the urease inhibitor of the present invention (OPC, Glory International Co., Ltd.) was determined. The catechins alone were compared with the urease inhibitory activity. In addition, (+)-epicatechin, (+)-catechin, and three kinds of dl-catechin were used as catechins. Urease inhibitors and catechins of the present invention were used at a concentration of 10 μg / ml, and the urease inhibition rate (%) was calculated for each. Table 2 shows the results.

[0034]

Table 2 Comparison of urease inhibitory activity between urease inhibitor of the present invention and catechins Urease inhibition rate (%) Urease inhibitor of the present invention 84.1 (+)-epicatechin 30.7 (+) -Catechin 23.8 dl-Catechin 26.9

From Table 2, it can be seen that the urease inhibition rate of catechins ((+)-epicatechin, (+) catechin, dl-catechin) was about 25 to 30%, whereas the inhibition rate of the urease inhibitor of the present invention was about 25 to 30%. Was well over 80%. As described in a) above, the composition of the urease inhibitor of the present invention is about 65% proanthocyanidin and contains only about 30% catechins. This strongly suggested the ability of proanthocyanidin to inhibit urease. In addition, it was found that the combination of proanthocyanidins and catechins exhibited stronger urease inhibitory activity.

Example 2 Antiulcer Effect of Urease Inhibitor of the Present Invention Using rats, the antiulcer effect of the urease inhibitor of the present invention was evaluated. As the urease inhibitor of the present invention, "OPC" (trade name) provided by Glory International Co., Ltd. was used in the same manner as in Example 1. In particular,
Wistar / ST male rats (body weight about 2
60 g, n = 3 to 4) were orally administered with the test drug, the urease inhibitor of the present invention, or cimetidine (Cimetidine: used as a positive control) (all prepared with physiological saline). The dose was 10 mg / kg (body weight) of cimetidine and 1 mg / k for the urease inhibitor of the present invention.
g (body weight), 3 mg / kg (body weight), 10 mg / kg
(Weight) in three stages. Thereafter, each rat was placed in a Tokyo University medicinal stress cage and immersed in water at 23 ° C. up to the xiphoid process of the rat. The time from administration of the test drug to immersion in water was about 10 minutes. Seven hours later, the rats were removed from the stress cage, the rats were killed with ether, and the stomach of each rat was removed and treated with formalin. Thereafter, the stomach is incised along the great bay, and the ulcer area (unit: mm ² ) generated in the glandular stomach is measured by a stereoscopic microscope, and the ulcer area per animal is represented by the ulcer index (unit: mm ² ).
And As a control, an experiment was similarly performed on rats to which saline was orally administered instead of the test drug (saline-administered group). The ulcer index between the saline administration group (negative control group) and the test drug administration group (test drug group) was compared, and the ulcer occurrence inhibition rate (unit%) was determined by the following formula. FIG. 6 shows the results. Ulceration inhibition rate (%) = (control group-test drug group) / control group × 100

FIG. 6 shows that the use of the urease inhibitor of the present invention can significantly suppress the development of ulcers, especially stress ulcers. The effect was comparable to or better than that of the known antiulcer drug cimetidine.

Example 3 Capsule; (Composition in 1 capsule) OPC 100 mg Lactose 103 mg Maize starch 75 mg Magnesium stearate 2 mg Total 280 mg Powder of the above formulation was uniformly mixed and passed through a 60 mesh sieve. 280 mg of the powder was placed in a No. 3 gelatin capsule to prepare a capsule.

Example 4 Tablets; (Composition in one tablet) OPC 52 mg Microcrystalline cellulose 49 mg Corn starch 48 mg Magnesium stearate 1 mg Total 150 mg 52 mg of OPC, 49 mg of microcrystalline cellulose, and a part of corn starch were mixed. Granulated by the method. After passing the obtained granules through a sieve, the remaining corn starch and magnesium stearate were added and mixed. Next, the obtained granules were subjected to 15 tablets using a tableting machine.
0 mg tablets were obtained.

Example 5 Cookies; (Composition in Cookie Dough) OPC 10mg Soft Flour 3500mg Butter 1750mg Sugar 1500mg Melted Egg 750mg 75 10mg 1750mg of Butter and 1500mg of Sugar are added, and 750mg of Egg is added, and 10mg of OPC When,
A mixture of 3500 mg of flour was added to make a cookie dough. The cookie dough was baked in a 170 ° C. oven to make cookies.

Example 6 Candy; (composition of one candy) OPC 5 mg starch syrup 600 mg sugar 5000 mg 56 05 mg Using each of the above-mentioned raw materials, candy was prepared according to a conventional method.

Example 7: Senbei (Composition in one dough for dough) OPC 10 mg Rice flour 5000 mg Water 5000 mg 100 10 mg Using the above-mentioned raw materials, a senbei was prepared according to a conventional method.

[0043]

EFFECT OF THE INVENTION The urease inhibitor of the present invention containing proanthocyanidin or catechins has a high urease inhibitory activity, and since each component is also present in edible plants, it can be used in living organisms. It has no adverse effects, low toxicity and high safety. For this reason, it can be used for the purpose of inhibiting the proliferation or eradication of H. pylori by inhibiting the urease activity of H. pylori present in the living body such as the gastrointestinal tract, and for the ulcer (eg, gastric ulcer) caused by H. pylori. Useful for treatment or prevention. As described above, the urease inhibitor of the present invention can be used as an active ingredient of a medicament and as a food additive for the above purpose. In addition, proanthocyanidin, or the urease inhibitor of the present invention containing a further catechins as a food composition, if it is ingested, pylori in the stomach is naturally inhibited from growing, and is eventually disinfected, In addition, ulcers caused by pylori can be prevented or treated.

The fluorescent labeling method of the present invention can accurately evaluate and measure urease activity even in the presence of a compound having a phenol structure, since there is no problem such as the reaction of the compound with a measurement reagent. . Therefore, the use of the fluorescent labeling method of the present invention makes it possible to screen and evaluate the urease inhibitory activity of a compound having a phenol structure for which the urease inhibitory activity could not be evaluated and measured by the conventional indophenol method.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing the effect of H. pylori infection on mucous membranes.

FIG. 2 is a diagram for explaining the measurement principle of an indophenol method which is a conventional method for measuring urease inhibitory activity.

FIG. 3 is a view for explaining the measurement principle of the fluorescent label method, which is a method for measuring urease inhibitory activity of the present invention.

FIG. 4: Urease inhibitor of the present invention (OP
C, Glory International Co., Ltd.).

FIG. 5 is a view showing a chemical structural formula of a positive control (positive control) compound.

FIG. 6 is a graph showing the effect of the urease inhibitor of the present invention on preventing stress ulcer in rats.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/04 A61K 31/00 631C 43/00 643D A61K 35/78 35/78 B C12N 9/99 C12N 9 / 99 G01N 33/15 G01N 33/15 Z // C07D 311/62 C07D 311/62 F term (reference) 4B018 LB01 LB08 MD15 ME08 ME11 4B035 LC06 LE05 LG04 LG31 LG35 LG37 LK19 LP02 LP21 4C062 FF03 FF56 4C086 AA01 A02A02A MA02 MA04 ZA68 ZB35 ZC20 ZC78 4C088 AB03 AC04 AC06 BA08 BA32 CA03 NA14 ZA68 ZB35 ZC20

Claims (18)

[Claims] 1. A urease inhibitor comprising proanthocyanidin. 2. The urease inhibitor according to claim 1, further comprising a catechin. 3. A proanthocyanidin content of 60 to 95% by weight.
3. The urease inhibitor according to claim 2, comprising 5 to 40% by weight of catechins and catechins. 4. The urease inhibitor according to claim 1, wherein the proanthocyanidins are derived from pine bark or grape seeds. 5. A medicament comprising the urease inhibitor according to any one of claims 1 to 4 as an active ingredient. 6. The medicament according to claim 5, which is used as a growth inhibitor or a disinfectant for Helicobacter pylori. 7. The medicament according to claim 5, which is used as an anti-ulcer agent. 8. A helicobacter, comprising the urease inhibitor according to claim 1.
A food additive used in foods for the purpose of inhibiting the growth or elimination of H. pylori in vivo. 9. A food additive containing the urease inhibitor according to any one of claims 1 to 4, which is used in food for the purpose of anti-ulcer. 10. A food composition comprising proanthocyanidin and having growth inhibitory or bacterial elimination properties against Helicobacter pylori. 11. The food composition according to claim 10, further comprising catechins. 12. The food composition according to claim 11, wherein the proportion of the proanthocyanidins and the catechins is 5 to 67% by weight of the catechins based on 100% by weight of the proanthocyanidins. 13. The proanthocyanidin is derived from pine bark or grape seeds.
3. The food composition according to any one of 2. 14. A food composition containing proanthocyanidins and having antiulcer properties. 15. The food composition according to claim 14, further comprising catechins. 16. The food composition according to claim 15, wherein the proportion of the proanthocyanidins and the catechins is 5 to 67% by weight of the catechins relative to 100% by weight of the proanthocyanidins. 17. The proanthocyanidin is derived from pine bark or grape seeds.
7. The food composition according to any one of 6. 18. A method for measuring urease activity, comprising producing ammonia by hydrolyzing urea with urease, and then reacting the produced ammonia with dansyl chloride to determine the amount of dansylamide produced.